Figure 1.

Generation of immortal and transformed human cell lines. (A) Immortalized (MRC-5^hTERT) and transformed (MRC-5^TSR) human cell lines were generated from embryonic lung fibroblasts MRC-5 by stepwise infection with retroviral particles driving the expression of hTERT, SV40 T-Ag and H-RAS^V12G. ‘Neo’, ‘Hygro’ and ‘Puro’ indicate drug resistance markers neomycin, hygromycin and puromycin, respectively, carried by the retroviral vectors. (B) Growth of MRC-5, MRC-5^hTERT and MRC-5^TSR cell lines measured as population doublings over 400 days in culture. The parental MRC-5 cell line enters senescence after ∼20 pd. (C) Telomerase activity detected by TRAP in cell extracts from MRC-5, MRC-5^hTERT and MRC-5^TSR cells. ‘Ctrl’ is a control telomerase-positive NCI-H520 lung cancer cell line. The ‘plus’ and ‘minus’ symbols indicate whether the extracts have been subjected to heat inactivation (HI). ‘pd’ represents population doublings. (D) A Western blot probed with anti-SV40 T-Ag antibodies shows stable expressed of T-Ag only in MRC-5^TSR cell. HDAC1 is a loading control. (E) MRC-5^TSR cells show elevated levels of H-RAS protein due to expression of exogenous H-RAS^V12G (see also Supplementary Figure S1D). α-Tubulin is a loading control.